J Bacteriol Virol.  2007 Sep;37(3):177-191. 10.4167/jbv.2007.37.3.177.

Studies on the Development of Viral Detection Markers for the Quality Control of Blood

• Kim Y
• Lee JH
• Kim Y

• KMID: 1513573
• DOI: http://doi.org/10.4167/jbv.2007.37.3.177

Abstract

According to the serological screening methods of antigen-antibody reaction such as ELISA, it has been known that the complete detection of viral infections of HBV, HCV, and HIV-1 viruses in the blood and blood related-products is not much reliable. Therefore, nucleic acid amplification testing methods (NAT) adopted to detect the small quantitative viral nucleic acids could support the basis of using and supplying the blood and its related products safely. This research work is basically designed to describe the simultaneous blood screening system by multiplex or duplex tests for detection of HBV, HCV, and HIV-1 viruses in the blood at one time with low price and labor. It is aimed at easy detection by using the conventional agarose gel electrophoresis. Thus, we tried to detect and identify the viral components in the blood sample according to their different size of PCR products. We decided a set of consensus sequences to recognize each viral DNA fragments after running the multiplex PCR in one tube. This was done by nested RT-PCR using two different RNA viral genomic templates followed by multiplex PCR with addition of viral DNA and their primers after purifying the viral genomic nucleic acids. Those specific primers could be used without any interference to amplify each viral genome in the blood samples. The sensitivities with different viral loads were evaluated on the agarose gel electrophoresis. Three different viral agents in the blood samples could be tested by this multiplex (RT)-PCR with three different primers.

Keyword

HBV; HCV; HIV-1; Multiplex (RT)-PCR; Blood sample

MeSH Terms

Antigen-Antibody Reactions
Consensus Sequence
DNA, Viral
Electrophoresis, Agar Gel
Enzyme-Linked Immunosorbent Assay
Genome, Viral
HIV-1
Mass Screening
Multiplex Polymerase Chain Reaction
Nucleic Acid Amplification Techniques
Nucleic Acids
Polymerase Chain Reaction
Quality Control*
RNA
Running
Viral Load
Viral Structures
DNA, Viral
Nucleic Acids
RNA

Figure

• 

  Figure 1. Detection limit of HBV (A), HCV (B), HIV-1 (C) DNA plasmid samples using PCR. M, 100 bp DNA ladder; Each lanes was half-diluted from 240 ng/ml (A), 240 ng/ml (B), 1,300 ng/ml (C). Positive PCR product were shown on the 336 bp size (A), 250 bp size (B), and 501 bp size (C), respectively.

• 

  Figure 2. (A) Detection limit of Multiplex PCR with combination of different plasmid NAs of HBV, HCV, and HIV-1 samples on the 1.4% agarose gel: at the lane 1, HBV (60 ng/ml) plus HCV (45 ng/ml) plus HIV-1 (325 ng/ml) were mixed with 1 μl of each into the reaction volume, and lane 2 was used with half of lane 1, lane 3 was used with half of lane 2, and so on; (B) Detection limit of multiplex PCR with labeled with 32P-dTTP was carried on the 5% Polyacrylamide gel: C, positive control; lane 1, 0.1 μg/ml of each plasmid DNAs, respectively; lanes 2∼8 were used with serial dilution of 10 fold, respectively. The upper bands are shown as HIV-specific DNA (501 bp), middle bands are shown as HBV-specific DNA (336 bp), and lower bands are shown as HCV-specific DNA (250 bp).

• 

  Figure 3. Reproducibility of (RT)-PCR methods HBV (A), HCV (B), HIV (C) DNA PCRs were performed three times by three different persons, respectively. 1, higher concentration than detection limit; 2, similar concentration of detection limit; 3, lower concentration than detection limit.

• 

  Figure 4. Multiplex PCR Amplification with each different plasmid DNA template (1, HBV; 2, HCV; 3, HIV-1), with two combined templates (4, HBV + HCV; 5, HBV + HIV-1; 6, HCV + HIV-1), and with three different templates together (7, HBV + HCV + HIV-1).

• 

  Figure 5. Twenty different sera positively diagnosed by ELISA tests were amplified by multiplex PCR with 6 different primers: M, 100 bp marker; NC, negative control sera without any template DNA; C, three different plasmid DNAs such as HBV (A), HCV (B), HIV-1 (C), were amplified by same PCR for the control; each other lane is shown as each different sera samples amplified by multiplex PCR methods.

• 

  Figure 6. Confirmation of the multiplex (RT)-PCR in one tube. Multiplex-PCR were performed with HBV-, HCV-positive sera, and sera containing HIV-1 plasmid DNA: Multiplex-PCR were performed with HBV-, HCV-positive sera, and HIV-1 plasmid: M, 100 bp size marker DNAs; lane 1, HBV-specific primer; lane 2, HCV-specific primer; lane 3, HIV-1 specific primer with HIV-1 plasmid DNA; lane 4, HBV and HCV specific primers; lane 5, HBV and HIV-1 specific primers; lane 6, HCV and HIV-1 specific primers; lanes 7 and 8, HBV, HCV, and HIV-1 specific primers were used, respectively. HBV sample number 10, HCV sample number 2 were used in the reaction. And also 1 μl of 1 μg/ml HIV-1 plasmid DNA was added into reaction

Reference

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